In the previous article, we examined how the complement systems recognize antigens and how they are activated through three pathways for host defense. Now, in this article, let's look in detail at the specific roles that those activated complements play in host defense. Their functions in the immune system can be broadly divided into three categories: (1) opsonization of the target pathogen cell surface (C3b, C5b), (2) recruitment and activation of immune cells by production of potent inflammatory anaphylatoxins (C3a, C5a), and (3) assembly of the Membrane Attack Complex (MAC) to induce lysis and death of target pathogens (C5b, C6–C9) .
1. Opsonins and opsonization: C3b, C4b
Complement fragments initiate by docking with antibodies (immune complexes) bound to pathogens or by binding to specific carbohydrates on glycoproteins in the pathogen's cell membrane, leading to the formation of C3 convertase. The alternative pathway also begins spontaneously and then produce C3 convertase. Interestingly, all these activities occur only on or very close to the surface of pathogen cells. In fact, this is a very fortunate phenomenon from the perspective of host defense. Many C3b and especially C5b fragments created through the cleavage by C3 convertase and C5 convertase stick closely to the surface of the pathogen cells. This phenomenon of coating the cell surface is called opsonization, and the attaching C3b and C5b are referred to as opsonins.
Think about it from the pathogen's perspective. It would be very difficult for it to move since its entire body would be surrounded by chunks of fragments. However, the purpose of opsonins is not to harass the pathogen. They serve as a preparatory step for phagocytes, such as macrophages and neutrophils, that will rush in to engulf them. It’s like putting a tag on delicious food, or bread crumbs attached to a piece of pork cutlet. This makes it look more appetizing. These immune cells express complement receptors (CR) that bind well to the complement proteins.
Let’s take a closer look at the mechanism of action of opsonin. All cell membranes carry a negative charge, which makes it difficult for two cells to get close. This means that the cell walls of pathogens and macrophages push each other away, making it challenging for the macrophages to engulf the pathogens. Opsonins resolve this repulsion by coating the surfaces of pathogens, which increases the speed at which macrophages can phagocytize them. Importantly, opsonins do not adhere to the host's healthy cells; they have evolved to recognize and distinguish between pathogenic molecules and damaged molecules. Opsonins accomplish this through the carbohydrate recognition domain (CRD), which binds to oligosaccharides on the surface of microorganisms.
Are host cells also targets of opsonization?
You might wonder whether C3b molecules opsonize host cells, including humans. Fortunately, they do not. The human body produces complement factor H, which serves as a defense mechanism for host cells. Factor H has a strong affinity for unique carbohydrate structures on host cells (primarily sialic acid) that are generally absent on pathogens. By recognizing and binding to these structures first, factor H intercepts C3b and prevents it from binding to host cells, ensuring that they do not become targets of the immune attack.
While we'll revisit complement inhibition in a later discussion, it's worth noting that the fluid form of factor H that does not bind to the cell membrane competes with factor B to bind to C3b in the alternative pathway. By first binding to C3b and forming C3bH, factor H effectively inhibits the formation of C3 convertase and prevents complement activation. Moreover, it aids in activating factor I to facilitate the breakdown of C3b into its inactive form.
The role of macrophages as Cleaners
Here, let’s briefly touch on one more important role of macrophages. Not only do macrophages consume foreign antigens, such as pathogens that invade from the outside, but they also act as cleaners, moving around to engulf debris left by damaged host tissues or cells that have undergone normal programmed cell death(apoptosis). By keeping our bodies clean and free of waste, they help maintain our health. If these tissue remnants are not removed in a timely manner, it could lead to autoimmune reactions. As mentioned in the first article on the complement system, complement molecules have various roles beyond immune activity. They also assist macrophages by opsonizing not only pathogens but also the self-materials that need to be cleared away. Personally, whenever I think of macrophages, I can't help but be reminded of No-Face from the animation movie, "Spirited Away"... 😊
Types of opsonins
Opsonins are associated with both innate and adaptive immunity. Antibodies (IgG, IgM), acute-phase reactive proteins (CRP), complement proteins (C3b, C4b), and various calcium-dependent lectins can all act as opsonins. The mannose-binding lectin (MBL), which belongs to the collectin family of lectins, along with ficolins and lung surfactant proteins A and D(SP-A and SP-D) present in the liquid that washes the epithelial surfaces of the lungs also function as opsonins. Among the surfactant proteins, SP-A and SP-D play crucial roles in protecting the host by inhibiting excessive inflammatory responses that could damage the lungs and disrupt gas exchange.[1] The epithelial surface of the lungs, which boasts a surface area comparable to a tennis court, is a delicate pathway constantly exposed to pathogens, allergens, and toxic gases. Therefore, it is essential to have thorough host defense mechanisms in place. To this end, alveolar macrophages and dendritic cells reside in the alveoli, continuously sampling and inspecting the inhaled air. When pathogens are detected, these proteins coat (opsonize) the surface of the pathogens, thus facilitating the phagocytosis of macrophages. SP-A and SP-D are structurally very similar to C1q of the classical pathway, as well as MBL, but they do not activate the complement system since they are not connected to MASP.
2. Inflammatory mediators: C5a > C3a > C4a
In terms of the cascade sequence, the fragments C4a, generated by C1s, C3a created by the C3 convertase, and C5a activated by the C5 convertase are all inflammatory mediators that activate immune cells and induce an inflammatory response. Interestingly, the intensity of the inflammatory response they generate increases as you move further down the cascade, with C5a being the most potent. In fact, C4a has a very minimal role in inducing inflammation and shows almost no inflammatory receptor activity, thus it is hardly considered an inflammatory mediator.
If C3a and C5a are produced in excess, they can lead to systemic allergic reactions similar to shock. This reaction is termed anaphylactic shock, for which C3a and C5a are also known as anaphylatoxins or anaphylactic peptides.
Inflammatory response
Local inflammatory response is a reaction aimed at directing immune cells in the bloodstream to migrate to the infected tissue. When the fragments C3a and C5a bind to GPCRs (G Protein-Coupled Receptors) on the surfaces of monocytes (eosinophils, neutrophils, basophils), macrophages, mast cells, and endothelial cells, it triggers a signaling cascade that leads to the secretion of cytokines such as IL-6 and TNF-α. The release of these cytokines causes contraction of the smooth muscles surrounding the blood vessels, resulting in dilation of the vessels, increased blood flow, and enhanced vascular permeability, allowing fluids to seep more easily from the vessels into the tissues. This movement of fluids leads to swelling in the affected area.
The C3a and C5a fragments also secrete chemokines like IL-8 (CXCL8) to attract and guide immune cells circulating in the blood to the site of infection. When immune cells recognize these chemokines, they move toward the endothelial layer of the blood vessels. The endothelial cells near the infected tissue help these immune cells adhere to the vessel walls by expressing adhesion molecules. When inflammatory signals bind to the GPCRs on the endothelial cells, a signaling cascade results in the expression of adhesive molecules such as P-selectin and E-selectin. For the small immune cells, the flowing blood may feel like a rushing river, and without the adhesive receptors pulling them in, they would simply be swept away. Subsequently, when endothelial cells contract, they create small gaps for the immune cells to squeeze through, allowing them to use amoeba-like shapes to push through into the tissues. This process is known as diapedesis (It seems that it has an etymology similar to that of the Jewish diaspora). This is well expressed in the figure below. Of course, remember that the size of immune cells is actually much smaller.
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Upon arriving in the tissues, neutrophils and macrophages attach to pathogens that are coated with opsonins like C3b and C5b and bind to complement receptors (CR). These immune cells then engulf the pathogens. Mast cells and basophils degranulate to release histamine to assist in removing the pathogens. The primary function of histamine is also to dilate blood vessels and increase their permeability. Anyone who has ever taken antihistamines knows what can happen when histamine levels are excessively high.
Histamine reaction
When you look at the reaction caused by histamine secreted from the granules of mast cells, you think of it as a very instinctive struggle or twitching to expel something that has entered from the outside. To eliminate foreign substances that have entered through the digestive system, the body increases gastric secretion and induces peristalsis, leading to vomiting or diarrhea. To expel foreign substances that have entered through the respiratory tract, the body produces excessive mucus and phlegm, provoking sneezes or coughs and narrowing the airways to prevent further intake of these substances. As the airways constrict and secretions increase, inhaled air becomes trapped in the lungs, making it very difficult to breathe, potentially leading to an asthma attack.
Additionally, to gather more effector cells, such as leukocytes and plasma proteins that are involved in immune functions, the blood vessels dilate and their permeability increases. When blood vessels dilate throughout the body, it can lead to serious situations. This is because increased vessel size can result in a rapid drop in blood pressure, causing hypotensive shock. As a result, if vital organs do not receive adequate blood supply and oxygen levels drop, it could lead to death. In severe cases, epinephrine is usually administered to relax the smooth muscles of the airways and reverse the effects of histamine, aiding in recovery
3. Membrane Attack Complex and Cell Lysis: C5b6789 Complex
Complement proteins can directly destroy pathogens by forming pores in their cell surfaces. The first step of MAC formation is when the C5 convertase cleaves C5, releasing C5b. The released C5b binds to nearby C6 to form the complex C5b6. When C7 attaches to this complex, it undergoes a structural transformation, exposing a hydrophobic region that gets inserted into the lipid bilayer of the pathogen’s cell. Subsequently, the C8 molecule also binds to the complex, exposing its hydrophobic region, which is likewise embedded in the lipid bilayer. Ultimately, around 10 to 19 C9 fragments begin to polymerize, finishing the formation of a pore approximately 10 nm in diameter. The Membrane Attack Complex (MAC) is now complete.
Organisms that maintain integrity and homeostasis through their cell membranes find it difficult to perform normal metabolic or physiological activities when holes are created in the cell membrane. Due to the punctured pores, an osmotic pressure difference arises between the inside and outside of the pathogen's cell. This leads to a continuous influx of water and electrolytes, while substances inside the cell may also leak out. In summary, the pathogen swells until it can no longer withstand the pressure, ultimately resulting in a disastrous rupture, a terrible end.
There are pathogens and bacteria that are particularly vulnerable to lysis due to the attack from the MAC. Herpes viruses, orthomyxoviruses that cause measles and mumps, paramyxoviruses such as influenza, and retroviruses are all enveloped viruses that are susceptible to complement-mediated lysis. Gram-positive bacteria with thick cell walls (peptidoglycan) can withstand these attacks because the complement fragments have difficulty penetrating the cell wall to reach the membranes beyond. In the case of Gram-negative bacteria with two cell membranes, it seems that the MAC limits its action to the area where the inner and outer membranes meet, attacking both membranes. Gram-negative bacteria like Neisseria meningitidis, which causes meningitis, are especially susceptible to MAC attacks.
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From innate immunity to adaptive immunity
We have examined the three main roles of the complement system. When pathogens begin their attack on the host, molecules such as MBL, C1q, C3b, and C4b bind to the antigens. These molecules then attach to the complement receptors on antigen-presenting cells, such as dendritic cells and macrophages, initiating the antigen recognition signaling process. Additionally, when antigen-presenting cells bind to anaphylatoxin complement molecules through anaphylatoxin receptors (C5aR), they secrete cytokines like IL-12, stimulating naive T cells to differentiate into Th1 cells. B cells that recognize the antigen-complement complex also start producing antibodies to help clear the pathogens.
An important aspect related to the role of complements is that they are actively involved in the immune system, contributing diversified defenses from the early stages of infection through to adaptive immunity. This emphasizes the complement system's comprehensive contribution to host defense.
[References]
[1] Immunomodulatory Roles of Surfactant Proteins A and D
doi: 10.1513/pats.200701-018AW. PMID: 17607008; PMCID: PMC2647627.
[Basic Readings]
Kuby Immunology. 8th New York: Macmillan Learning, 2019. Text. MLA Style. Punt, Jenni, Stranford, Sharon A, Jones, Patricia P, Owen, Judith A.
Janeway's immunobiology. Kenneth Murphy, Janeway Jr., Paul Travers, Walport Sir. 9th Edition, New York, Garland Science/Taylor & Francis Group, LLC, [2016]
Fundamental Immunology 5th edition (August 2003): William E. Paul (Editor). Philadelphia: Lippincott Williams & Wilkins, c2003.
Roitt's Essential Immunology, Thirteenth Edition. Peter J. Delves, Seamus J. Martin, Dennis R. Burton, and Ivan M. Roitt. Published 2017 by John Wiley & Sons, Ltd
